As discussed in our published overview of the study, we enrolled 50,884 US and Puerto Rican women who were between the ages of 35 and 74 and had a sister with breast cancer but did not have breast cancer themselves when they joined the study between 2003 and 2009. At enrollment, data on potential risk factors and current health status were collected using computer assisted telephone interviews and mailed questionnaires. Blood, urine, and environmental samples were collected in a home visit and banked for future use in nested studies. More than 3,000 Sister Study participants have reported a diagnosis of invasive or in situ breast cancer. The cohort is tracked annually for changes in vital status and major health outcomes. Detailed follow-up questionnaires on health outcomes, environmental and lifestyle exposures, and special topics are completed every 2-3 years. We retrieve medical records and tumor tissue for those who develop cancer or other conditions of interest. In 2014-15 we repeated the collection of biological (including toenail clippings) and environmental samples from women diagnosed with breast cancer since enrollment and a random sample of the cohort. Of approximately 3,800 participants invited, samples were collected from 2,436 (63%) including 1,229 women who had been diagnosed with breast cancer. These paired repeat samples have allowed us to explore changes in biomarkers and exposures over time and in relation to breast cancer diagnosis and treatment. Several of the publications listed below made use of those twice-collected biospecimens. Comparing the concentrations of 16 trace elements from toenails collected at baseline and in 2014-15, we found minimal evidence for age effects but found that levels generally had decreased over calendar time, particularly for cadmium, chromium and lead. Breast cancer and ovarian cancer cases through 2014 and a random sample of the cohort have been genotyped as part of the multi-study Oncoarray project. Through this project, Sister Study and Two Sister Study data have been included in several collaborative analyses, including two recent transcriptome-wide association studies. One (Mavaddat et al.) developed a polygenic risk score and related it to the tumor subtypes, and one (Fachel, et al.) did fine-mapping of 150 genes related to breast cancer. In other collaborative work (Choudhury, et al.), a polygenic risk score was validated using 14 prospective cohort studies. In another consortial project headed up by my former postdoc (O'Brien, et al.), now a Staff Scientist in the Epidemiology Branch, the use of perineal talc and douching were studied in relation to risk of uterine cancer. A separate consortial project looked at the same exposures in relation to risk of ovarian cancer. A consortial project headed up by a former NIEHS postdoc (Nichols, et al.) looked at breast cancer risk in relation to recent childbirth and found evidence for increased risk in the few years after delivering a baby. We also previously generated data on 450,000 CpGs for the non-Hispanic white women in the genotyping sample, with plans to evaluate methylation patterns in relation to risk factors of interest. The first such analysis to be published was an analysis of vitamin D, DNA methylation and breast cancer. We also have used epigenetic data to apply recently developed methylation-based biologic clocks and learned that evident acceleration in biologic aging is predictive of risk of breast cancer (Kresovich, et al., in press at JNCI). In separate work (white, et al.), we found evidence that an epigenome-based measure of accelerated biologic aging is related to air pollution. We had previously observed an inverse association between high levels of serum 25-hydroxyvitamin D and breast cancer risk. Following-up on this work, we reported that single nucleotide polymorphisms in vitamin D-related genes may modify vitamin D-breast cancer associations. In a genome-wide association study, we identified two loci associated with serum 25-hydroxyvitamin D levels. Using data from the Two Sister Study, metal exposures (as assessed in toenails) were not associated with young-onset breast cancer. In work soon to be submitted (Diaz, et al.), we used the Two Sister Study data to assess very early life experiences in relation to risk of young-onset (under age 50) breast cancer and found evidence that women born to a preeclamptic pregnancy were at increased risk, particularly when adjustment is made in the logistic model for whether they had later gestated a preeclamptic pregnancy themselves. In other work soon to be submitted (von Holle, et al.) we found evidence of familial clustering in the age at onset of breast cancer, a finding with implications for both screening and etiology. Iron is a growth factor that is essential to life and also promotes lipid peroxidation and the formation of reactive oxygen species (ROS). There is reason to think that iron status can influence the risk of breast cancer. High meat diets have repeatedly been shown to be associated with increased risk, and consumption of heme iron (mainly derived from consumption of red meat) may drive this association. Under a contract with the University of Minnesota, we have now measured serum iron, ferritin (a protein that stores and binds iron, sequestering it from infectious agents) and transferrin saturation (another marker for stored iron) in a case-cohort sample nested within the Sister Study. Analysis is beginning for that project. Some of our work has been related to non-cancer endpoints. In one such project (Park, et al.), we found evidence that exposure to light at night, e.g. from a television left on, contributes to obesity (cross-sectionally) and to weight gain (prospectively), even after adjustment for measures of sleep duration and quality. In work currently under review, exposure to mercury, as assessed in toenails, was found to be related to the neurodegenerative disease, ALS. Also (2 papers by Basso, et al.), advanced age of the mother at the time of birth could contribute to life-long childlessness and (in the daughters who do conceive) to reduced risk of preeclampsia .